Part:BBa_K2500000
Heme-Deleted Bacterioferritin (M52H)
Bacterioferritins are bacterial iron storage proteins. It has been shown that overexpression of bacterioferritin in E. coli Nissle 1917 can lead to a visible contrast change in MRI, which allows for visualization of the bacteria. We used a heme-deletion mutant as it does not decrease contrast change.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Usage and Biology
Ferritins are iron storage proteins found in different species. Through ferroxidase, they convert iron from its toxic, ferrous state (Fe2+) into a non-toxic, ferric state (Fe3+). When present in its ferric state, iron acts as a paramagnetic agent and causes a shortening of the transversal relaxation (T2) in MRI. Depending on the concentration of iron, this can lead to a visible change in contrast.1
Bacterioferritin is one of the three forms of ferritin-like proteins found in bacteria. It has been shown that overexpression of bacterioferritin in E. coli Nissle 1917 can lead to a visible contrast change in MRI, which allows for visualization of the bacteria. Additionally, by site directed mutagenesis, resulting in a heme-deleted version of bacterioferritin, it was shown that the absence of heme does not influence the contrast change.2
For this part, a decrease in signal intensity was observed for all bacteria grown in iron-supplemented medium, independent of bacterioferritin, probably due to presence of various inherent bacterial iron-storage proteins. However, an additional significant drop in the signal was observed in samples where bacterioferritin overexpression was induced as expected. This result proves the usability of bacterioferritin as an MRI contrast agent in vitro and confirms the potential to use it as an in vivo reporter of tumor sensing.
Design
Bacterioferritin was integrated into our system to serve as an MRI reporter, able to point out locations inside the body colonized by bacteria. When iron is present in the medium, overexpression of bacterioferritin leads to accumulation of iron, which causes a shortening of the T2 relaxation time, seen as a drop in the signal intensity (i.e. darker area on an MRI T2 scan).1
To test the consequences of bacterioferritin overexpression in an MRI scanner, we transformed E. coli Nissle 1917 with a plasmid containing an AHL-inducible promoter (pLux) that controls the expression of both a green fluorescent protein (GFP) and bacterioferritin (Figure 1).
Characterization
AHL-induced bacterioferrtin production
First, we determined the concentration of AHL needed for full induction of the system. To do this, we measured changes in fluorescence caused by different concentrations of AHL used for induction (Figure 2). Second, we performed an SDS-PAGE analysis that confirmed that bacterioferritin was indeed expressed along with GFP after induction with the appropriate concentration of AHL (Figure 3).
Consequences of bacterioferritin overexpression of MRI signals
Finally, we grew the bacteria in an iron-supplemented medium (150 µM of ferric citrate in M9 minimal medium) to observe the consequences of bacterioferritin overexpression on the MRI signal. Biological triplicates of E. coli Nissle transformed with the heme-deleted bacterioferritin were grown in four different experimental conditions (with and without induction and with and without iron supplementation) and imaged in a 4.7 T small animal MRI scanner. A bacterioferritin-expressing E. coli Top 10 (T7lacO-bfr) was used to compare the effect of the heme-deleted bacterioferritin against the wild-type bacterioferritin. Additionally, a bfr-knockout E. coli K-12 from the Keio collection was tested.
All bacteria grown in iron-supplemented medium showed a drop in the T2 signal intensity, independent of induction of bacterioferritin expression. However, once induced, they experienced an additional drop in the signal, as predicted (Figure 3). The drop in the signal was more pronounced for the heme-deleted verion of bacterioferritin as compared to the wild-type version.
A basal T2 level was determined as the mean value of the T2 relaxation rate of the bacteria grown without iron. To calculate the change in the T2 relaxation rate, the basal T2 level was subtracted from the values obtained for the bacteria grown in the presence of iron (Figure 5). A higher values indicates a bigger change in the signal.
All the bacteria were resuspended and imaged in PBS, after washing of the culture medium. To test if any unwashed iron could mask the signal, the change in the T2 relaxation rate was compared in pure PBS versus PBS supplemented with iron. Moreover, a bfr-knockout was imaged to see how much the endogenous bacterioferritin contributes to the signal when the bacteria are grown in the presence of iron. The results showed that the free iron in the medium only slightly changes the signal and should not interfere with the measurements. On the other hand, the bfr-knockout strain showed the same behaviour as the wild-type bacteria, suggesting that other iron-storage systems present in the bacteria contribute to iron uptake significantly (Figure 6). The differences in absolute values of the signal changes might be explained by the fact that different strains of E. coli were imaged. T7lacO-bfr is Top 10, pLux-bfr M52L is Nissle, while the bfr-knockout is K-12.
References
1. Cohen, Batya et al. “Ferritin as an Endogenous MRI Reporter for Noninvasive Imaging of Gene Expression in C6 Glioma Tumors.” Neoplasia (New York, N.Y.) 7.2 (2005): 109–117. Print.
2. Hill, Philip J., et al. "Magnetic resonance imaging of tumors colonized with bacterial ferritin-expressing Escherichia coli." PLoS One 6.10 (2011): e25409.
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